Design and formulation of polyplexes based on pluronic-polyethyleneimine conjugates for gene transfer

Previously, we reported the evaluation of several polyplex-based gene delivery systems with respect to their effectiveness, toxicity, and cell type dependence in vitro. One system, P123-g-PEI(2K), a cationic graft block copolymer, is of particular interest as it has been demonstrated to successfully deliver genetic material to murine liver following systemic delivery [Nguyen, H. K., Lemieux, P., Vinogradov, S. V., Gebhart, C. L., Guerin, N., Paradis, G., Bronich, T. K., Alakhov, V. Y., and Kabanov, A. V. (2000) Evaluation of Polyether-Polyethyleneimine Graft Copolymers as Gene Transfer Agents. Gene Ther. 7, 126-138 (1)]. The P123-g-PEI(2K) system requires nonmodified Pluronic P123 as an excipient to stabilize the dispersion. The purpose of the current work was to more closely characterize this system, to assess the role of each component of the system to the overall transfection process. We evaluated particle size, stability, and resistance to nuclease degradation. In addition, cellular uptake and localization of plasmid, as well as transgene expression, were evaluated following in vitro transfection of prostate cancer cells (PC-3) with various individual components of the system. Nonmodified Pluronic alone did not significantly enhance DNA uptake, transgene expression, or DNase protection. Therefore, we conclude that nonmodified Pluronic acted primarily by optimizing the size of the polyplex. Furthermore, though this system displays several characteristics thought desirable of a nonviral gene delivery system, these studies did discriminate a potential limitation of this system for in vivo applications, namely, the insufficient level of protection of plasmid DNA from nuclease degradation. This may limit the effective dose delivered, as well as limiting the effective circulation time. These studies provide vital information that will guide modification of this system to enhance the current in vivo profile.     

Differential expression of alternative oxidase genes in maize mitochondrial mutants

We have examined the expression of three alternative oxidase (aox) genes in two types of maize mitochondrial mutants. Nonchromosomal stripe (NCS) mutants carry mitochondrial DNA deletions that affect subunits of respiratory complexes and show constitutively defective growth. Cytoplasmic male-sterile (CMS) mutants have mitochondrial DNA rearrangements, but they are impaired for mitochondrial function only during anther development. In contrast to normal plants, which have very low levels of AOX, NCS mutants exhibit high expression of aox genes in all nonphotosynthetic tissues tested. The expression pattern is specific for each type of mitochondrial lesion: the NADH dehydrogenase-defective NCS2 mutant has high expression of aox2, whereas the cytochrome oxidase-defective NCS6 mutant predominantly expresses aox3. Similarly, aox2 and aox3 can be induced differentially in normal maize seedlings by specific inhibitors of these two respiratory complexes. Translation-defective NCS4 plants show induction of both aox2 and aox3. AOX2 and AOX3 proteins differ in their ability to be regulated by reversible dimerization. CMS mutants show relatively high levels of aox2 mRNAs in young tassels but none in ear shoots. Significant expression of aox1 is detected only in NCS and CMS tassels. The induction pattern of maize aox genes could serve as a selective marker for diverse mitochondrial defects.     

Globin and linker sequences of the giant extracellular hemoglobin from the leech Macrobdella decora

A detailed electrospray ionization mass spectrometric study of the 3.5-MDa hexagonal bilayer hemoglobin (HBL Hb) from the pond leech Macrobdella decora has shown it to consist of at least six 17-kDa globin chains, of which two are monomeric and the remaining four occur as disulfide-bonded heterodimers, and three 24-kDa nonglobin linker chains (Weber et al., J. Mol. Biol. 251: 703–720, 1995). The cDNA sequences of the five major constituent chains, globin chains IIA, IIB, B, and C and linker chain L1, are reported here. The globins and linkers share 30%–50% and 20%–30% identity, respectively, with other annelid sequences. Furthermore, IIB and C align with strain A of annelid sequences, whereas IIA and B align with the strain B sequences. Although chains B and C are monomeric, chains IIA and IIB form the main disulfide-bonded dimer. They also have some unusual features: the distal His (E7) is replaced by Phe in IIA, and the highly conserved CD1Phe is replaced by Leu in IIB. In spite of these unusual features, the functional properties of Macrobdella Hb are comparable to those of other HBL Hbs. A phylogenetic analysis of the globin sequences from Macrobdella, the polychaete Tylorrhynchus, the oligochaete Lumbricus, and the vestimentiferan Lamellibrachia, indicates that the two strains originated by gene duplication followed by additional duplication of each of the two strains. The mutation rate of the linkers appeared to be faster than that of the globin chains. The phylogenetic trees constructed using the Maximum Likelihood, Neighbor-Joining and Fitch methods showed the Macrobdella globin sequences to be closest to Lumbricus, in agreement with a view of annelid evolution in which the divergence of the polychaetes occurred before the divergence of the leeches from oligochaetes.     

The structure of the carbohydrate backbone of the core-lipid A region of the lipopolysaccharide from Proteus vulgaris serotype O25

The following structure of the lipid A-core region of the lipopolysaccharide (LPS) from Proteus vulgaris serotype O25 was determined by using NMR and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of LPS, of the products of alkaline deacylation of the LPS, and of the products of LPS deamination: [structure: see text] Terminal residues of beta-GlcNAc and beta-Kdo (indicated by bold italics) are present alternatively in approximately 3:2 amount, leaving no unsubstituted beta-Gal. All sugars are in the pyranose form, alpha-Hep is the residue of L-glycero-alpha-D-manno-Hep, alpha-DDHep is the residue of D-glycero-alpha-D-manno-Hep.     

Combinatorics of giant hexagonal bilayer hemoglobins

The paper discusses combinatorial and probabilistic models allowing to characterize various aspects of spacial symmetry and structural heterogeneity of the giant hexagonal bilayer hemoglobins (HBL Hb). Linker-dodecamer configurations of HBL are described for two and four linker types (occurring in the two most studied HBL Hb of Arenicola and Lumbricus, respectively), and the most probable configurations are found. It is shown that, for HBL with marked dodecamers, the number of 'normal-marked' pairs of dodecamers in homological position follows a binomial distribution. The group of symmetries of the dodecamer substructure of HBL is identified with the dihedral group D6. Under natural symmetry assumptions, the total dipole moment of the dodecamer substructure of HBL is shown to be zero. Biological implications of the mathematical findings are discussed.     

The breeding productivity of dark-bellied brent geese and curlew sandpipers in relation to changes in the numbers of arctic foxes and lemmings on the Taimyr Peninsula, Siberia —

There were about three-year cycles in the populations of arctic foxes, and the breeding productivities of brent geese and curlew sandpipers on the Taimyr Peninsula, Russia, The populations of arctic foxes and lemmings changed in synchrony. The breeding productivities of the birds tended to be good when the arctic foxes were increasing in numbers and poor when the arctic foxes were decreasing. There was a negative relationship between arctic fox numbers (or occupied lairs) and the breeding productivity of brent geese in the following year. Although there was evidence of wide-spread synchrony In the lemming cycle across the Taimyr Peninsula, some localities showed differences, However, such sites would still have been influenced by the general pattern of fox abundance in the typical tundra zone of the Taimyr Peninsula, where most of the arctic foxes breed and from which extensive movements of foxes occur after a decline in lemming numbers. The results support a prey-switching hypothesis (also known as the alternative prey hypothesis) whereby arctic foxes, and other predators, feed largely on lemmings when these are abundant or increasing, but switch to birds when the lemming population is small or declining. The relationships between arctic foxes, lemmings and brent geese may be further influenced by snowny owls which create fox-exclusion zones around their nests, thus providing safe nesting areas for the geese.     

INTROGRESSION IN THE DROSOPHILA SUBOBSCURA—D. MADEIRENSIS SISTER SPECIES: EVIDENCE OF GENE FLOW IN NUCLEAR GENES DESPITE MITOCHONDRIAL DIFFERENTIATION

Species hybridization, and thus the potential for gene flow, was once viewed as reproductive mistake. However, recent analysis based on large datasets and newly developed models suggest that gene exchange is not as rare as originally suspected. To investigate the history and speciation of the closely related species Drosophila subobscura, D. madeirensis, and D. guanche, we obtained polymorphism and divergence data for 26 regions throughout the genome, including the Y chromosome and mitochondrial DNA. We found that the D. subobscura X/autosome ratio of silent nucleotide diversity is significantly smaller than the 0.75 expected under neutrality. This pattern, if held genomewide, may reflect a faster accumulation of beneficial mutations on the X chromosome than on autosomes. We also detected evidence of gene flow in autosomal regions, while sex chromosomes remain distinct. This is consistent with the large X effect on hybrid male sterility seen in this system and the presence of two X chromosome inversions fixed between species. Overall, our data conform to chromosomal speciation models in which rearrangements are proposed to serve as gene flow barriers. Contrary to other observations in Drosophila, the mitochondrial genome appears resilient to gene flow in the presence of nuclear exchange.